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Diversity of Bacterial Populations on the Tongue Dorsa of Patients with Halitosis and Healthy Patients

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The primary purpose of the present study was to compare the microbial profiles of the tongue dorsa of healthy subjects and subjects with halitosis by using culture-independent molecular methods. Our overall goal was to determine the bacterial diversity on the surface of the tongue dorsum as part of our ongoing efforts to identify all cultivable and not-yet-cultivated species of the oral cavity. Tongue dorsum scrapings were analyzed from healthy subjects with no complaints of halitosis and subjects with halitosis, defined as an organoleptic score of 2 or more and volatile sulfur compound levels greater than 200 ppb. 16S rRNA genes from DNA isolated from tongue dorsum scrapings were amplified by PCR with universally conserved bacterial primers and cloned into Escherichia coli. Typically, 50 to 100 clones were analyzed from each subject. Fifty-one strains isolated from the tongue dorsa of healthy subjects were also analyzed. Partial sequences of approximately 500 bases of cloned inserts from the 16S rRNA genes of isolates were compared with sequences of known species or phylotypes to determine species identity or closest relatives. Nearly complete sequences of about 1,500 bases were obtained for potentially novel species or phylotypes. In an analysis of approximately 750 clones, 92 different bacterial species were identified. About half of the clones were identified as phylotypes, of which 29 were novel to the tongue microbiota. Fifty-one of the 92 species or phylotypes were detected in more than one subject. Those species most associated with healthy subjects were Streptococcus salivarius, Rothia mucilaginosa, and an uncharacterized species of Eubacterium (strain FTB41). Streptococcus salivarius was the predominant species in healthy subjects, as it represented 12 to 40% of the total clones analyzed from each healthy subject. Overall, the predominant microbiota on the tongue dorsa of healthy subjects was different from that on the tongue dorsa of subjects with halitosis. Those species most associated with halitosis were Atopobium parvulum, a phylotype (clone BS095) of Dialister, Eubacterium sulci, a phylotype (clone DR034) of the uncultivated phylum TM7, Solobacterium moorei, and a phylotype (clone BW009) of STREPTOCOCCUS: On the basis of our ongoing efforts to obtain full 16S rRNA sequences for all cultivable and not-yet-cultivated species that colonize the oral cavity, there are now over 600 species.
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JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2003, p. 558–563 Vol. 41, No. 2
0095-1137/03/$08.000 DOI: 10.1128/JCM.41.2.558–563.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Diversity of Bacterial Populations on the Tongue Dorsa of Patients
with Halitosis and Healthy Patients
C. E. Kazor,
1
P. M. Mitchell,
2
A. M. Lee,
2
L. N. Stokes,
2
W. J. Loesche,
1
F. E. Dewhirst,
2,3
and B. J. Paster
2,3
*
Department of Microbiology and Immunology, School of Medicine, University of Michigan, Ann Arbor, Michigan,
1
and
Department of Molecular Genetics, The Forsyth Institute,
2
and Department of Oral and Developmental Biology,
Harvard School of Dental Medicine,
3
Boston, Massachusetts
Received 5 August 2002/Returned for modification 9 November 2002/Accepted 21 November 2002
The primary purpose of the present study was to compare the microbial profiles of the tongue dorsa of
healthy subjects and subjects with halitosis by using culture-independent molecular methods. Our overall goal
was to determine the bacterial diversity on the surface of the tongue dorsum as part of our ongoing efforts to
identify all cultivable and not-yet-cultivated species of the oral cavity. Tongue dorsum scrapings were analyzed
from healthy subjects with no complaints of halitosis and subjects with halitosis, defined as an organoleptic
score of 2 or more and volatile sulfur compound levels greater than 200 ppb. 16S rRNA genes from DNA
isolated from tongue dorsum scrapings were amplified by PCR with universally conserved bacterial primers
and cloned into Escherichia coli. Typically, 50 to 100 clones were analyzed from each subject. Fifty-one strains
isolated from the tongue dorsa of healthy subjects were also analyzed. Partial sequences of approximately 500
bases of cloned inserts from the 16S rRNA genes of isolates were compared with sequences of known species
or phylotypes to determine species identity or closest relatives. Nearly complete sequences of about 1,500 bases
were obtained for potentially novel species or phylotypes. In an analysis of approximately 750 clones, 92
different bacterial species were identified. About half of the clones were identified as phylotypes, of which 29
were novel to the tongue microbiota. Fifty-one of the 92 species or phylotypes were detected in more than one
subject. Those species most associated with healthy subjects were Streptococcus salivarius, Rothia mucilaginosa,
and an uncharacterized species of Eubacterium (strain FTB41). Streptococcus salivarius was the predominant
species in healthy subjects, as it represented 12 to 40% of the total clones analyzed from each healthy subject.
Overall, the predominant microbiota on the tongue dorsa of healthy subjects was different from that on the
tongue dorsa of subjects with halitosis. Those species most associated with halitosis were Atopobium parvulum,
a phylotype (clone BS095) of Dialister, Eubacterium sulci, a phylotype (clone DR034) of the uncultivated phylum
TM7, Solobacterium moorei, and a phylotype (clone BW009) of Streptococcus. On the basis of our ongoing efforts
to obtain full 16S rRNA sequences for all cultivable and not-yet-cultivated species that colonize the oral cavity,
there are now over 600 species.
Halitosis, or oral malodor, is a common complaint of up to
one-third of the general population and a large concern for the
many individuals whom it affects (6, 19). Halitosis can arise
from a variety of sources including the sinuses, gastrointestinal
tract, ingested food, lungs, and, most frequently, the oral cav-
ity. Oral production of malodorous substances is most com-
monly associated with by-products of bacterial metabolic deg-
radation and occurs on oral surfaces, in periodontal pockets,
and especially on the dorsal tongue surface. These products
result from microbial fermentation of proteins, peptides, and
mucins found in saliva, blood, gingival crevicular fluid, lysed
neutrophils, desquamated epithelial cells, and any residual
food retained on the oral surfaces (16). The most conspicuous
malodorous compounds are termed volatile sulfur compounds
(VSCs), with hydrogen sulfide, methyl mercaptan, and di-
methyl sulfide accounting for roughly 90% of the VSCs (32).
Many oral bacteria, especially gram-negative anaerobic species
found in the subgingival plaque, produce a diverse array of
malodorous compounds as by-products of their metabolism
including VSCs and short-chain organic acids such as valeric
acid, butyric acid, putrescine, and skatole (16). Species that
produce such malodorous compounds include Treponema den-
ticola, Porphyromonas gingivalis, Prevotella intermedia, Tan-
nerella forsythensis, Porphyromonas endodontalis, and Eubacte-
rium species (25, 26).
Halitosis has been correlated with the presence and severity
of periodontal disease and by the amount of coating on the
tongue (3, 20, 21). Various methods of detecting and quanti-
fying oral odor have been proposed, including organoleptic
odor rating schemes (smelling the breath) (27, 29, 35) and
analytical techniques involving gas chromatography, mass
spectrometry, and cryo-osmoscopy. Rosenberg and colleagues
(28) have reported on the use of a portable sulfide monitor
called a Halimeter (Interscan, Chatsworth, Calif.) to quanti-
tate the levels of VSCs in mouth breath and have shown that
these levels significantly correlate with the measurements
made by organoleptic odor rating schemes. In individuals with
oral malodor, tongue-coating samples have been shown to
hydrolyze N-benzoyl-
DL-arginine-2-naphthylamide (BANA)
(2, 4, 11, 21). Since the BANA test detects an arginine hydro-
lase produced by proteolytic bacteria, this test provides addi-
* Corresponding author. Mailing address: The Forsyth Institute, 140
Fenway, Boston, MA 02115. Phone: (617) 456-7716. Fax: (617) 456-
7737. E-mail: bpaster@forsyth.org.
558
tional information on the bacterial ora associated with mal-
odor.
Effective treatments of oral malodor consist of reducing the
bacterial load on the tongue and teeth through twice-daily
tooth brushing with uoride toothpaste and daily tongue de-
bridement with a toothbrush or other mechanical device, alone
or in combination with the use of antimicrobial mouth rinses
such as chlorhexidine (4, 11, 33, 36).
Although the bacteria of the tongue have been implicated as
a major source of odor production in subjects with halitosis,
the bacterial composition of the tongue is still not well char-
acterized. Studies of cultivable tongue microbiota have been
limited by the difculties of in vitro growth techniques, the low
percentage of recovery of total organisms, and the inadequacy
of microbial identication (7, 10). For example, Kazor et al.
(10) were able to recover only up to 30% of the viable micro-
bial count using a growth medium supplemented with human
blood and saliva. These ndings suggest that much of the
tongue microbiota has not yet been cultivated, necessitating
the use of molecular approaches to better characterize the
tongue microora.
Using culture-independent molecular methods, we had pre-
viously detected over 500 species or phylotypes in the subgin-
gival plaque of healthy subjects and subjects with periodontal
diseases (22), dental plaque in children with rampant caries
(1), and noma (24). Other investigators have used similar tech-
niques to determine the bacterial diversity of saliva (31), den-
toalveolar abscesses (5), and subgingival plaque of a subject
with gingivitis (12). The purpose of this study was to determine
the bacterial diversity on the tongue dorsum and to compare
the predominant bacteria (including not-yet-cultivated species)
that are present on the surface of the tongue dorsa of subjects
with and without oral malodor.
MATERIALS AND METHODS
Subject population. Halitosis in healthy adult subjects with self-reported ha-
litosis (n 6) was conrmed by an organoleptic rating of 2 or more and VSC
measurements greater than 200 ppb (Table 1). BANA hydrolysis (BANA Test;
OraTec Corp, Manassas, Va.) was also determined (Table 1), since positive
scores by the BANA test have previously been associated with a component of
oral malodor (2, 4, 11, 21). Five healthy subjects without evidence of halitosis
were used as controls.
Sample collection. Samples were collected by scraping the tongue surface from
the vallate papilla area to the anterior tongue border with a sterile wooden
tongue depressor. All tongue scrapings were used directly for clonal analysis as
described below. Strains were isolated from some samples on either ETSA
medium (enriched Trypticase soy agar to which hemin, menadione, nitrate, and
lactate were added to support growth of oral species) or MM10 medium (1/10
dilution of ETSA medium) (17).
Sample lysis. Pure cultures or tongue scrapings were directly suspended in 50
l of 50 mM Tris buffer (pH 7.6)1 mM EDTA (pH 8)0.5% Tween 20. Pro-
teinase K (200 g/ml) was added. The samples were then heated at 55°Cfor2h.
Proteinase K was inactivated by heating at 95°C for 5 min.
Amplication of 16S rRNA cistrons by PCR and purication of PCR products.
The 16S rRNA genes (rDNAs) were amplied under standard conditions with a
universal primer set (forward primer, 5-GAG AGT TTG ATY MTG GCT
CAG; reverse primer, 5- GAA GGA GGT GWT CCA RCC GCA) (22).
Primers were synthesized commercially (Operon Technologies, Alameda, Calif.).
PCR was performed in thin-walled tubes with a Perkin-Elmer 9700 thermocycler.
One microliter of the DNA template was added to a reaction mixture (nal
volume, 50 l) containing 20 pmol of each primer, 40 nmol of deoxynucleoside
triphosphates, and1UofTaq 2000 polymerase (Stratagene, La Jolla, Calif.) in
buffer containing Taqstart antibody (Sigma Chemical Co.). In a hot-start proto-
col, the samples were preheated at 95°C for 8 min, followed by amplication
under the following conditions: denaturation at 95°C for 45 s, annealing at 60°C
45 s, and elongation for 1.5 min, with an additional 5 s for each cycle. A total of
30 cycles were performed; this was then followed by a nal elongation step at
72°C for 10 min. The results of PCR amplication were examined by electro-
phoresis in a 1% agarose gel. DNA was stained with ethidium bromide and
visualized under short-wavelength UV light.
Cloning procedures. Cloning of PCR-amplied DNA was performed with the
TOPO TA cloning kit (Invitrogen, San Diego, Calif.) according to the instruc-
tions of the manufacturer. Transformation was done with competent Escherichia
coli TOP10 cells provided by the manufacturer. The transformed cells were then
plated onto Luria-Bertani agar plates supplemented with kanamycin, and the
plates were incubated overnight at 37°C. The colonies were then placed into 40
l of 10 mM Tris. One microliter was used as the template to determine the
correct sizes of the inserts in a PCR with an M13 (40) forward primer and an
M13 reverse primer. The sizes of the inserts (approximately 1,500 bp) were
determined by PCR with anking vector primers, followed by electrophoresis on
a 1% agarose gel. Prior to sequencing of the fragments, the PCR-amplied 16S
rDNA fragments were puried and concentrated with Microcon 100 (Amicon),
followed by use of the QIAquick PCR purication kit (Qiagen).
16S rRNA sequencing. Puried DNA from the PCR was sequenced with an
ABI Prism cycle sequencing kit (BigDye Terminator Cycle Sequencing kit with
AmpliTaq DNA Polymerase FS; Perkin-Elmer). The primers used for sequenc-
ing have been reported previously (22). Quarter dye chemistry was used with 80
M primers and 1.5 l of PCR product in a nal volume of 20 l. Cycle
sequencing was performed with an ABI 9700 instrument, with 25 cycles of
denaturation 96°C for 10 s, annealing, and extension at 60°C for 4 min. The
sequencing reactions were run on an ABI 377 DNA sequencer.
16S rRNA sequencing and data analysis of unrecognized inserts. A total of
741 clones with the insert of the correct size of approximately 1,500 bases were
analyzed (typically, 50 to 100 per subject). In addition, 51 strains from healthy
subjects without halitosis were analyzed. A sequence of approximately 500 bases
was obtained rst to determine identity or approximate phylogenetic position.
Full sequences of about 1,500 bases were obtained by using ve to six additional
sequencing primers (22) for those species deemed novel. For identication of
closest relatives, the sequences of the unrecognized inserts were compared to the
16S rRNA gene sequences of over 4,000 microorganisms in our database and
over 50,000 sequences in the Ribosomal Database Project (18) and GenBank
databases. Programs for data entry, editing, sequence alignment, secondary
structure comparison, similarity matrix generation, and phylogenetic tree con-
struction were written by F. E. Dewhirst (23). The similarity matrices were
corrected for multiple base changes at single positions by the method of Jukes
and Cantor (9). Similarity matrices were constructed from the aligned sequences
by using only those sequence positions for which data were available for 90% of
the strains tested. Phylogenetic trees were constructed by the neighbor-joining
method of Saitou and Nei (30). TREECON, a software package for the Mi-
crosoft Windows environment, was used for the construction and drawing of
evolutionary trees (34).
We are aware of the potential creation of 16S rDNA chimera molecules during
the PCR (14). The percentage of chimeric inserts in 16S rRNA libraries ranged
from 1 to 15%. Chimeric sequences were identied by using the Chimera check
program of the Ribosomal Database Project, treeing analysis, or base signature
TABLE 1. Clinical parameters of study population
Odor status
Subject
clone
Organoleptic
score
a
Volatile sulfur
compound
concn (ppb)
b
Tongue
coating
BANA test
result for
tongue
Malodor M1 3 350 ⫹⫹/
M2 4 411 ⫹⫺
M3 3 452 ⫹⫹/
M4 4 642 ⫹⫹/
M5 4 346 ⫹⫺
M6 4 749 ⫹⫹
Healthy H1 1 87 ⫺⫺
H2 2 160 ⫹⫹/
H3 2 144 ⫹⫺
H4 2 113 ⫺⫺
H5 2 132 ⫺⫹/
a
A score of 2 is associated with malodor.
b
A level of 200 ppb is associated with malodor (Halimeter; Interscan Cor
-
poration).
VOL. 41, 2003 TONGUE BACTERIA IN HALITOSIS AND HEALTHY PATIENTS 559
560 KAZOR ET AL. J. CLIN.MICROBIOL.
analysis. Species identications of the chimeras were obtained, but the partial
sequences were not included in the phylogenetic analysis for tree construction.
Nucleotide sequence accession numbers. The complete 16S rDNA sequences
of clones representing novel phylotypes dened in this study, the sequences of
known species not reported previously, and published sequences are available for
electronic retrieval from the EMBL, GenBank, and DDBJ nucleotide sequence
databases under the accession numbers shown in Fig. 1.
RESULTS AND DISCUSSION
A phylogenetic tree of the prevalent species and phylotypes
detected on the surface of the tongue dorsum for each of the
subjects is shown in Fig. 1. Collectively, the overall bacterial
diversity of the tongue dorsum is striking: 92 different bacterial
taxa or phylotypes belonging to six bacterial phyla. Only 38, or
about 40%, of the total number were identied as known
species. Consequently, about 60% of the total were identied
as phylotypes. As shown in Fig. 1, 29 of these phylotypes were
unique to the tongue dorsum, in that they were not found from
the sequence analysis of over 6,000 clones from other oral sites,
including the subgingival plaque of healthy subjects and sub-
jects with, periodontitis, acute necrotizing ulcerative gingivitis,
and refractory periodontitis (22); the supragingival plaque of
children with rampant caries (1); or advanced noma lesions
(24); nor were they found on or in crevicular epithelial cells of
healthy subjects and subjects with periodontitis (13). In our
ongoing studies, we have detected over 300 novel phylotypes
and 200 known species in oral sites. At present, we estimate
that over 700 bacterial species are present in the oral cavity,
over half of which we cannot presently cultivate. At the time of
this publication, a list of 630 species or phylotypes of the oral
cavity was compiled. The creation of a website is in progress;
however, an updated version of this list can be obtained from
the corresponding author.
The number of species or phylotypes that were detected in
each subject ranged from 12 to 29, with 16 to 21 species or
phylotypes in tongue samples from subjects without malodor
and from 12 to 29 species or phylotypes in tongue samples
from subjects with malodor (Table 2). The bacterial proles for
each of these subjects are depicted in the colored columns of
boxes in Fig. 1. Those species most associated with health were
Streptococcus salivarius, Rothia mucilaginosa (Stomatococcus
mucilaginosus), and an uncharacterized, cultivable species of
Eubacterium (strain FTB41) (Fig. 1 and Table 2). The 15 most
prevalent species or phylotypes are listed in Table 2, where
they comprise 60 to 85% of the total clones in subjects without
malodor and 20 to 88% of the total clones in subjects with
malodor. A prevalent species is dened as a species that was
detected in at least three subjects. It is noteworthy that S.
salivarius was by far the most predominant species detected in
healthy subjects: in one subject (subject H1), S. salivarius rep-
resented more than 40% of the detectable species. In contrast,
S. salivarius was detected in only one of the subjects with
halitosis and was detected at very low levels.
Those species most associated with halitosis were Atopobium
parvulum, Eubacterium sulci, Fusobacterium periodonticum,a
phylotype (clone BS095) of Dialister, a phylotype (clone
TABLE 2. Percentage of prevalent species or phylotypes on tongue dorsum
Species or phylotype
% Clones from:
Healthy subjects (no. of clones analyzed) Halitosis subjects (no. of clones analyzed)
H1 (102) H2 (51) H3 (68) H4 (65) H5 (76) M1 (50) M2 (56) M3 (46) M4 (81) M5 (78) M6 (47)
Atopobium parvulum 36 4851
a
Cryptobacterium curtum 19
Dialister sp. clone BS095 4 8 2
Eubacterium sulci 24 46
Fusobacterium periodonticum 5 24
Granulicatella adiacens 8 21 5 14 42774
Neisseria flavescens 47 24
Rothia mucilaginosa 64310 54
Streptococcus infantis 1 29654 420 1
Streptococcus parasanguis 27 18 7 32 8 18 92794
Streptococcus pneumoniae 1 10 3
Streptococcus salivarius 41 24 26 12 12 56
Streptococcus sp. clone BW009 4119
Streptococcus strain HalT4-E3 1 2 24 86
Veillonella parvula/V. dispar 36311 22 2 10 1
No. of species detected 21 22 18 16 16 16 29 15 26 28 12
% of total detected 85 60 76 76 69 68 20 71 68 59 88
a
Values in boldface indicate that the species comprised at least 10% of the tongue microbiota.
FIG. 1. Phylogenetic tree of bacterial species or phylotypes of six phyla identied from the tongue dorsa of healthy subjects and subjects with
halitosis. The information presented includes bacterial species or phylotype clone and sequence accession numbers. Novel phylotypes are dened
as those taxa that are 98.5 to 99% similar in sequence comparisons to their closest relatives. Species or phylotypes detected only on the tongue
dorsum and not at any other oral site are highlighted in red. Color-coordinated characters indicate health status category or some other site at
which each species was identied. Bar, 5% difference in nucleotide sequence. ANUG, acute necrotizing ulcerative gingivitis; NUP, necrotizing
ulcerative periodontitis.
VOL. 41, 2003 TONGUE BACTERIA IN HALITOSIS AND HEALTHY PATIENTS 561
BW009) of Streptococcus, a phylotype (clone DR034) of the
uncultivated phylum TM7 (8), and Solobacterium moorei (Fig.
1 and Table 2). Note that in most of the samples, several
species or phylotypes represented a signicant proportion of
the total (Table 2). Although some species were not detected
in all subjects, they were the predominant species in one or
more samples. For example, Cryptobacterium curtum was de-
tected in only one of the samples from a subject with halitosis,
but it represented about 20% of the clones analyzed in that
subject. Other species, such as Granulicatella (Abiotrophia)
adiacens, Streptococcus parasanguis, Streptococcus infantis, and
Veillonella spp., were commonly detected in most of the sam-
ples (Table 2).
The tongue dorsum harbors a highly diverse, yet character-
istic, bacterial population. In healthy subjects, S. salivarius was
by far the predominant species. In contrast, S. salivarius was
typically absent from subjects with halitosis. Although bacteria
other than S. salivarius appeared to be associated with halito-
sis, it is not known if they are directly involved in oral malodor.
Cultural studies have associated R. mucilaginosa with malodor
(7) and S. salivarius and Veillonella parvula or Veillonella dispar
as common healthy tongue organisms (C. E. Kazor and W. J.
Loesche, unpublished data).
On the basis of our sequence analyses, the tongue dorsum
possesses a unique microbiota: about one-third of the bacterial
population was found only on the tongue and not in or on the
surfaces of other oral sites. However, a sample of sufcient size
(e.g., in a large clinical trial) is necessary to provide the power
to detect differences in microbial compositions to identify
more precisely those species that are associated with halitosis
and health. Such studies will be accomplished by using 16S
rRNA-based oligonucleotide probes in checkerboard DNA-
DNA hybridization assays (1) or eventually by using oligonu-
cleotide microarrays.
It has been suggested that the majority of cases of oral
malodor are due to bacterial proteolytic activity in the mouth,
such as might be measured by the BANA test (16). Since
known BANA test-positive oral species typically found in sub-
gingival plaque, i.e., P. gingivalis, T. denticola, T. forsythensis,
and various Capnocytophaga species, were not detected (15),
other tongue bacterial species are likely responsible for the
BANA reaction of the tongue coating. Now that we have
identied additional predominant bacterial species present on
the tongue dorsa of individuals with halitosis, it would be of
interest to examine the ability of these other cultivable species
to hydrolyze the BANA substrate and to produce VSCs and
other by-products that may contribute to the clinical presen-
tation of malodor.
ACKNOWLEDGMENTS
This study was supported by NIH grants DE12465 and DE11443
from the National Institute of Dental and Craniofacial Research.
REFERENCES
1. Becker, M. R., B. J. Paster, E. J. Leys, M. L. Moeschberger, S. G. Kenyon,
J. L. Galvin, S. K. Boches, F. E. Dewhirst, and A. L. Griffen. 2002. Molecular
analysis of bacterial species associated with early childhood caries. J. Clin.
Microbiol. 40:10011009.
2. Bosy, A., G. V. Kulkarni, M. Rosenberg, and C. A. McCulloch. 1994. Rela-
tionship of oral malodor to periodontitis: evidence of independence in dis-
crete subpopulations. J. Periodontol. 65:3746.
3. De Boever, E. H., M. DeUzeda, and W. J. Loesche. 1994. Relationship
between volatile sulfur compounds, BANA-hydrolyzing bacteria and gingival
health in patients with and without complaints of oral malodor. J. Clin. Dent.
4:114119.
4. De Boever, E. H., and W. J. Loesche. 1995. Assessing the contribution of
anaerobic microora of the tongue to oral malodor J. Am. Dent. Assoc.
126:13841393.
5. Dymock, D., A. J. Weightman, C. Scully, and W. G. Wade. 1996. Molecular
analysis of microora associated with dentoalveolar abscess. J. Clin. Micro-
biol. 34:537542.
6. Frexinos, J., P. Denis, H. Allemand, S. Allouche, F. Los, and G. Bonnelye.
1998. Descriptive study of digestive functional symptoms in the French
general population. Gastroenterol. Clin. Biol. 22:785791.
7. Hartley, M. G., M. A. El-Maaytah, C. McKenzie, and J. Greenman. 1996.
The tongue microbiota of low odour and malodorous individuals. Microb.
Ecol. Health Dis. 9:215223.
8. Hugenholtz, P., C. Pitulle, K. L. Hershberger, and N. R. Pace. 1998. Novel
division level bacterial diversity in a Yellowstone hot spring. J. Bacteriol.
180:366376.
9. Jukes, T. H., and C. R. Cantor. 1969. Evolution of protein molecules, p.
21132. In H. N. Munro (ed.), Mammalian protein metabolism, vol. 3.
Academic Press, Inc., New York, N.Y.
10. Kazor, C. E., J. R. Flowers, J. Stoll, and W. J. Loesche. 1999. Oral malodor:
dening the normal tongue ora. J. Dent. Res. 78:421.
11. Kozlovsky, A., D. Gordon, I. Gelernter, W. J. Loesche, and M. Rosenberg.
1994. Correlation between the BANA test and oral malodor parameters. J.
Dent. Res. 73:10361042.
12. Kroes, I., P. W. Lepp, and D. A. Relman. 1999. Bacterial diversity within the
human subgingival crevice. Proc. Natl. Acad. Sci. USA 96:1454714552.
13. Levin, I. M., C. N. Lau, S. S. Socransky, A. D. Haffajee, L. Martin, J. L.
Galvin, S. K. Boches, B. J. Paster, and F. E. Dewhirst. 1999. Cultivable and
uncultivable species on or in gingival epithelial cells. J. Dent. Res. 78:453.
14. Liesack, W., H. Weyland, and E. Stackebrandt. 1991. Potential risk of gene
amplication by PCR as determined by 16S rDNA analysis of a mixed-
culture of strict barophilic bacteria. Microb. Ecol. 21:191198.
15. Loesche, W. J., W. A. Bretz, D. Kerschensteiner, J. A. Stoll, S. S. Socransky,
P. P. Hujoel, and D. E. Lopatin. 1990. Development of a diagnostic test for
anaerobic periodontal infections based on plaque hydrolysis of benzoyl-
DL-
arginine naphthylamide. J. Clin. Microbiol. 28:15511559.
16. Loesche, W. J., and C. E. Kazor. 2002. Microbiology and treatment of
halitosis. Periodontology 2000 28:256279.
17. Loesche, W. J., and S. A. Syed. 1973. The predominant cultivable ora of
carious plaque and carious dentine. Caries Res. 7:201216.
18. Maidak, B. L., J. R. Cole, T. G. Lilburn, C. T. Parker, Jr., P. R. Saxman, R. J.
Farris, G. M. Garrity, G. J. Olsen, T. M. Schmidt, and J. M. Tiedje. 2001.
The RDP-II (Ribosomal Database Project). Nucleic Acids Res. 29:173174.
19. Miyazaki, H., S. Sakao, K. Yasuhiro, and T. Tadamichi. 1995. Oral malodor
in the general population of Japan, p. 119136. In M. Rosenberg (ed.), Bad
breath research perspectives. Ramot Publishing, Tel Aviv, Israel.
20. Morita, M., and H. L. Wang. 2001. Association between oral malodor and
adult periodontitis: a review. J. Clin. Periodontol. 28:813819.
21. Morita, M., and H. L. Wang. 2001. Relationship between sulcular sulde
levels and oral malodor in subjects with periodontal disease. J. Periodontol.
72:7984.
22. Paster, B. J., S. K. Boches, J. L. Galvin, R. E. Ericson, C. N. Lau, V. A.
Levanos, A. Sahasrabudhe, and F. E. Dewhirst. 2001. Bacterial diversity in
human subgingival plaque. J. Bacteriol. 183:37703783.
23. Paster, B. J., and F. E. Dewhirst. 1988. Phylogeny of campylobacters,
wolinellas, Bacteroides gracilis, and Bacteroides ureolyticus by 16S rRNA
sequencing. Int. J. Syst. Bacteriol. 38:5662.
24. Paster, B. J., W. A. Falkler, Jr., C. O. Enwonwu, E. O. Idigbe, K. O. Savage,
V. A. Levanos, M. A. Tamer, R. L. Ericson, C. N. Lau, and F. E. Dewhirst.
2002. Predominant bacterial species and novel phylotypes in advanced noma
lesions. J. Clin. Microbiol. 40:21872191.
25. Persson, S., R. Claesson, and J. Carlsson. 1989. The capacity of subgingival
species to produce volatile sulfur compounds in human serum. Oral Micro-
biol. Immunol. 4:169172.
26. Persson, S., M. B. Edlund, R. Claesson, and J. Carlsson. 1990. The forma-
tion of hydrogen sulde and methylmercaptan by oral bacteria. Oral Micro-
biol. Immunol. 5:195201.
27. Pitts, G., R. Pianotti, T. W. Feary, J. McGuiness, and T. Masurat. 1981. The
in vivo effects of an antiseptic mouthwash on odor-producing microorgan-
isms. J. Dent. Res. 60:18911896.
28. Rosenberg, M., G. V. Kulkarni, A. Bosy, and C. A. McCulloch. 1991. Re-
producibility and sensitivity of oral malodor measurements with a portable
sulde monitor. J. Dent. Res. 70:14361440.
29. Rosenberg, M., I. Septon, I. Eli, R. Bar-Ness, I. Gelernter, S. Brenner, and
J. Gabbay. 1991. Halitosis measurement by an industrial sulphide monitor. J.
Periodontol. 62:487489.
30. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method
for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406425.
31. Sakamoto, M., M. Umeda, I. Ishikawa, and Y. Benno. 2000. Comparison of
562 KAZOR ET AL. J. CLIN.MICROBIOL.
the oral bacterial ora in saliva from a healthy subject and two periodontitis
patients by sequence analysis of 16S rDNA libraries. Microbiol. Immunol.
44:643652.
32. Tonzetich, J. 1971. Direct gas chromatographic analysis of sulphur com-
pounds in mouth air in man. Arch. Oral Biol. 16:587597.
33. Tonzetich, J., and S. K. Ng. 1976. Reduction of malodor by oral cleansing
procedures. Oral Surg. Oral Med. Oral Pathol. 42:172181.
34. Van de Peer, Y., and R. De Wachter. 1994. TREECON for Windows: a
software package for the construction and drawing of evolutionary trees for
the Microsoft Windows environment. Comput. Appl. Biosci. 10:569570.
35. Yaegaki, K., and J. M. Coil. 2000. Examination, classication, and treatment
of halitosis; clinical perspectives. J. Can. Dent. Assoc. 66:257261.
36. Yaegaki, K., and K. Sanada. 1992. Biochemical and clinical factors inuenc-
ing oral malodor in periodontal patients. J. Periodontol. 63:783789.
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Supplementary resources (35)

... For these reasons, the most common microorganisms are Bacteroides, Bifidobacterium, Streptococcus, Enterobacteriaceae, Enterococcus, Clostridium, Lactobacillus, and Ruminococcus [8] ( Table 1). This type of complex mutualistic interaction between these microorganisms and their host appears to have evolved over thousands of years [2]. ...
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... clone DP009, Streptococcus australis, Granulicatella adiacens, Gemella haemolysans, and Veillonella spp [30,31]. Streptococcus salivarius, Rothiamucila ginosa (Stomatococcus mucilaginosus), and an unidentified, cultivable sp of Eubacterium (strain FTB41) are the tongue sp most associated with health [32]. Streptococcus mitis, Streptococcus mitis biovar 2, Streptococcus sp. ...
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... Analyser av avskrap fra tungeryggen hos pasienter med og uten halitose viser stor forskjell med hensyn til bakterieinnhold. Pasienter uten halitose har overvekt av den bacteriocin-produserende Streptococcus salivarius til forskjell fra halitosepasienter, som viser et mylder av anaerobe bakteriearter (29). Kliniske forsøk over noen dager tyder på at tilførsel av S. salivarius som probiotika i sugetabletter reduserer de flyktige, svovelholdige forbindelser som er fremherskende ved halitose (30). ...
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Organoleptic and gas chromatographic methods were employed to establish the threshold of odor objectionability of methylmercaptan and hydrogen sulfide and to assess the relative effectiveness of different oral hygiene measures to reduce the malodor to acceptable levels. The study showed that methylmercaptan and hydrogen sulfide concentrations below 0.5 ng. and 1.5 ng., respectively, are considered nonobjectionable. Gas chromatographic analyses indicate that these concentrations were exceeded in the early morning mouth air samples of approximately 50 per cent of the adult population studied. In these instances, methylmercaptan and hydrogen sulfide occurred in sufficiently high concentrations to account for the malodor. Brushing studies suggest that the early morning malodor arising from the oral cavity can be controlled by proper oral hygiene. The tongue was the major source of both offending compounds in the persons studied. Methods that involved cleansing of the dorsoposterior surface of the tongue caused the most pronounced reductions of both compounds. Since methylmercaptan was found to be more objectionable and to exhibit a lower threshold of objectionability, it was more difficult to reduce to acceptable levels.